The reaction of physiological significance catalyzed by carbonic anhydrase (CA) is the hydration of carbon dioxide: CO2 + H20 _ HCO3- + H*. This catalysis requires attack on CO2 by zinc-bound hydroxide followed by rate-limiting proton transfers from the active site to solution to regenerate the zinc-bound hydroxide. The efficient isozymes of the animal CA's utilize His64 as an intramolecular proton shuttle; this residue accepts protons from the zinc-bound water through a network of hydrogen-bonded waters at a turnover rate of 106 s1 and transfers it to solution. The unifying goal of this proposal is to expand our use of the carbonic anhydrases to understand rate-limiting proton transfer steps in a way that can be extended to other proteins. A concurrent goal is to apply Marcus rate theory both to understand the proton transfers in carbonic anhydrase and to elucidate the significance of the parameters of the Marcus theory for proton transfer in an enzyme or protein. We will use site-specific mutagenesis and chemical modification to place proton transfer groups at strategic locations in isozymes of human CA. This provides us a range of catalytic activities, geometries, and active-site environments. We wilt also utilize exogenous proton donors from solution to expand the work to intermolecular proton transfer. Stopped-flow spectrophotometry and 180 exchange between CO2 and water measured by mass spectrometry will be used to obtain rate constants for inter- and intramolecular proton transfer. Crystal structures of important mutants will be determined by X-ray and neutron diffraction. We will determine specifically how distances, location, and environment in the active site influence the rate of proton transfer. We will apply Marcus rate theory to determine and interpret the intrinsic energy barriers and thermodynamic components for the proton transfers and relate them to the structural and chemical features of the CA active site.